Just as the elucidation of the structure of DNA provided valuable insights into the way the genetic material was replicated and information encoded, we believe that the solving of the structure of the eukaryotic chromosome will provide analogous types of insights into the way genes are regulated and chromosomes are replicated in higher organisms. We therefore wish to continue our analysis of the structure of the eukaryotic chromosome. This analysis will focus on three primary points: (1) the general structure of bulk chromatin, (2) the structure of chromatin that is actively being transcribed and (3) the in vivo mechanism of assembly of newly-replicated DNA into chromatin. Our investigation will involve several very sensitive techniques that we and others have developed over the past few years: (1) enzyme digestion of chromatin using specific, purified nucleases, proteases, and restriction enzymes, (2) affinity labeling of histones using such reagents as I-125 and fluorescamine, (3) cross-linking histones with formaldehyde, dimethyl superimidate, and tetranitro-methane and (4) analyzing the transcription by E. coli RNA polymerase or endogenous RNA polymerase with specific probes for specific base sequences. We believe that the clue to our understanding of eukaryotic gene regulation lies in the understanding of how histones, possibly in conjunction with other nuclear elements, generate a large number of sub-chromosomal units and that this requirement to form many types of interactions can explain why the amino acid sequences of the majority of histones are so rigidly conserved. H. Weintraub and M. Groudine, Chromosomal Subunits in Active Genes Have An Altered Conformation. Science 193, 848-856 (1976). H. Weintraub, A. Worcel and B. Alberts, A Model for Chromatin Based Upon Two Symmetrically Paired Half Nucleosomes. Cell 9, 409-417 (1976).